DNA polymerases typically catalyze nucleic acid synthesis using polynucleotide templates and employ Watson-Crick base pairing interactions between the template-based-nucleotide and incoming nucleotides which bind at the polymerase active site. DNA polymerases are useful in a variety of biological applications, including DNA sequencing applications.
Certain single molecule DNA sequencing methods encompass two steps, relying on nucleotide transient-binding to the polymerase, instead of nucleotide incorporation (e.g., Vander Horn, et al., U.S. Ser. No. 61/184,774; 61/242,763; and 61/295,533). The first step includes transiently-binding an incoming nucleotide (e.g., labeled nucleotide) with a polymerase under conditions which inhibit incorporation of the bound nucleotide, and the identifying the transiently-bound nucleotide. The second step includes incorporating a single nucleotide (e.g., terminator nucleotide) so as to translocate the polymerase to the next position on the DNA template. The transiently-bound nucleotide can be replaced with the nucleotide to be incorporated.
It is desirable to perform the first and second steps under moderate temperature conditions (e.g., room temperature) with one type of DNA polymerase. It is also desirable to use a DNA polymerase which binds incorporating incoming nucleotides that are complementary to the template-based-nucleotide. It is also desirable to use a DNA polymerase which exhibits increased transient-binding duration (without nucleotide incorporation) for the incoming nucleotides, to increase the binding duration of a labeled nucleotide to a polymerase, so as to increase detection and identity of the bound nucleotide.
However, existing polymerases cannot be used to perform the first and second steps because they lack certain properties. For example, many DNA polymerases do not selectively bind labeled nucleotides. Similarly, many DNA polymerases do not efficiently incorporate terminator nucleotides. Some DNA polymerases can exhibit short nucleotide binding duration, or can catalyze nucleotide incorporation under conditions when transient nucleotide binding is desired. Additionally, some DNA polymerases can exhibit undesirable behaviors such as binding non-complementary incoming nucleotides, or incorporating non-complementary incoming nucleotides.
Thus, existing polymerases offer limited utility for conducting certain two-step, single molecule sequencing methods. These and other desirable properties can be enhanced via modifying and selecting a DNA polymerase. Provided herein are mutant RB69 DNA polymerases which are useful for conducting these two-step DNA sequencing methods. In some embodiments, these mutant DNA polymerases can selectively and transiently bind labeled nucleotides. In some embodiments, the mutant DNA polymerases can incorporate terminator nucleotides.